PROJECT SUMMARY: This proposal investigates the mechanism(s) downstream of delta that promote differentiation. The Notch-Delta signaling pathway is a conserved regulator of differentiation in animals. Improper Notch- Delta signaling leads to defects in organogenesis, cell fate specification, maintenance of stem cells, and can result in aggressive tumor growth in cancers. The majority of research investigating Notch- Delta signaling focuses on activation of Notch, and its role in promoting maintenance of a proliferative undifferentiated stem-like cell fate. However, Delta is also activated, but little is known about what events occur downstream of Delta activation. Multiple studies suggest that Delta activation leads to cellular differentiation. Release and nuclear localization of the Delta intracellular domain appear to be a key factors to promote differentiation. Preliminary data during development of the sea anemone Nematostella vectensis argue that the intracellular domain of Delta (DeltaICD) localizes to the nucleus, and that DeltaICD is necessary and sufficient to promote neural differentiation. We identified ~80 putative down stream targets of Delta. This proposal takes advantage of these observations to investigate the role of Delta during differentiation. In aim1, the requirement for the nuclear localization of the intracellular domain of Delta to promote differentiation is tested. We will generate an antibody against the DeltaICD, generate mutants lacking the DeltaICD, and use misexpression of nuclear localized or nuclear excluded Deltas to determine if endogenous DeltaICD localizes to the nucleus and if nuclear localization is required to promote expression of neural differentiation markers. Aim2 focuses on determining which putative Delta targets most likely promote differentiation. To achieve this goal we first establish which targets require the DeltaICD using qPCR analysis to determine genes with opposite changes in expression in delta knockdowns compared to the change in expression observed in animals with hyperactivated Delta. Second, we use mRNA in situ expression approaches to determine which genes are expressed in a pattern consistent with an upstream regulator of differentiation. This work will confirm that Delta promotes differentiation by regulating expression of differentiation markers, and it will identify key genes for futures studies that will result in a detailed mechanistic understanding of cellular differentiation downstream of Delta. These data will not only improve our understanding of how normal development occurs, but will also inform us about the biology of aggressive cancers and perhaps offer novel therapeutic approaches to treat them.